Genetic alterations associated with cancer

ABSTRACT

The present invention provides new probes for the detection of chromosomal alterations associated with cancer, particularly ovarian cancer. The probes bind selectively with target nucleic acid sequences at 3q26.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of co-pending application U.S. Ser. No.08/837,046, filed Apr. 5, 1997, now U.S. Pat. No. 6,110,673 which is acontinuation-in-part of U.S. Ser. No. 08/783,729, filed Jan. 16 1997,now U.S. Pat. No. 6,277,563 each of which is incorporated herein byreference.

BACKGROUND OF THE INVENTION

Molecular genetic mechanisms responsible for the development andprogression of many cancers remain largely unknown. Identification ofsites of frequent and recurring allelic deletion or gain is a first steptoward identifying some of the important genes involved in the malignantprocess. Previous studies in retinoblastoma (Friend, et al. Nature,323:643-6 (1986)) and other cancers (Cawthon, et al., Cell, 62:193-201(1990); Baker, et al., Science, 244:217-21 (1989); Shuin, et al., CancerRes, 54:2832-5 (1994)) have amply demonstrated that definition ofregional chromosomal deletions occurring in the genomes of human tumorscan serve as useful diagnostic markers for disease and are an importantinitial step towards identification of critical genes. Similarly,regions of common chromosomal gain have been associated withamplification of specific genes (Visakorpi, et al., Nature Genetics,9:401-6 (1995)).

Comparative genomic hybridization (CGH) is a relatively new moleculartechnique used to screen DNA from tumors for regional chromosomalalterations (Kallioniemi, et al., Science, 258:818-21 (1992) and WO93/18186). Unlike microsatellite or Southern analysis allelotypingstudies, which typically sample far less than 0.1% of the total genome,a significant advantage of CGH is that all chromosome arms are scannedfor losses and gains. Moreover, because CGH does not rely on naturallyoccurring polymorphisms, all regions are informative, whereaspolymorphism-based techniques are limited by homozygous (uninformative)alleles among a fraction of tumors studied at every locus.

Increases in copy number in the long arm of chromosome 3, in particular3q25-3qter, has been associated with cancer. Increases in copy number inthis area have been seen not only in ovarian tumors (Iwabuchi et al.,Cancer Research 55:6172-8180 (1995) but also in brain tumors, head andneck cancer, lung cancer, ductal breast cancer, renal cell and otherurinary tract cancers, and cervical cancer. Ried et al., GenesChromosomes Cancer 15:234-45 (1996); Yeatman et al. Clin Exp Metastasis14:246-52 (1996); Brzoska et al., Cancer Res 15:3055-9 (1995); Ried etal., Cancer Res 54:1801-6 (1994); and Speicher et al., Cancer Res55:1010-3 (1995).

The identification of narrower regions of genetic alteration or genesassociated with cancers such as ovarian cancer would be extremely usefulin the early diagnosis or prognosis of these diseases. The presentinvention addresses these and other needs.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for detectinggenetic alterations correlated with cancer. The invention can be used todetect alterations in a 2 MB region at 3q26.3 that are associated with anumber of cancers. Examples include ovarian cancer, brain cancer, lungcancer, head and neck tumors, renal cell and other urinary tumors,cervical cancer, and ductal breast cancer. The invention is particularlyuseful for detecting alterations associated with ovarian cancer.

The methods comprise contacting a nucleic acid sample from a patientwith a probe which binds selectively to a target nucleic acid sequenceon 3q26.3 correlated with cancer. In particular, the invention providessequences from genes encoding the catalytic subunit ofphosphatidylinositol kinase type 3 (PIK3CA) or the glucose transporter,GLUT2. The probes of the invention are contacted with the sample underconditions in which the probe binds selectively with the target nucleicacid sequence to form a hybridization complex. The formation of thehybridization complex is then detected. Typically, the number of regionsof hybridization are counted. Abnormalities are detected as increasesabove normal in the regions of hybridization. In some embodiments, themethods of the invention further comprise detection of amplifications at19q13.1-13.2. This region includes AKT2, a putative oncogene.

Alternatively, sample DNA from the patient can be fluorescently labeledand competitively hybridized against fluorescently labeled normal DNA tonormal lymphocyte metaphases or to arrays of nucleic acid moleculeswhich map to 3q26.3. Alterations in DNA copy number in the sample DNAare then detected as increases in sample DNA as compared to normal DNAat the 3q26.3 region.

Definitions

A “nucleic acid sample” as used herein refers to a sample comprising DNAin a form suitable for hybridization to a probes of the invention. Thenucleic acid may be total genomic DNA, total mRNA, genomic DNA or mRNAfrom particular chromosomes, or selected sequences (e.g. particularpromoters, genes, amplification or restriction fragments, cDNA, etc.)within particular cancer-associated amplifications. The nucleic acidsample may be extracted from particular cells or tissues. The tissuesample from which the nucleic acid sample is prepared is typically takenfrom a patient suspected of having the disease associated with theamplification being detected. The sample may be prepared such thatindividual nucleic acids remain substantially intact and typicallycomprises interphase nuclei prepared according to standard techniques. A“nucleic acid sample” as used herein may also refer to a substantiallyintact condensed chromosome (e.g. a metaphase chromosome). Such acondensed chromosome is suitable for use as a hybridization target in insitu hybridization techniques (e.g. FISH). The particular usage of theterm “nucleic acid sample” (whether as extracted nucleic acid or intactmetaphase chromosome) will be readily apparent to one of skill in theart from the context in which the term is used. For instance, thenucleic acid sample can be a tissue or cell sample prepared for standardin situ hybridization methods described below. The sample is preparedsuch that individual chromosomes remain substantially intact andtypically comprises metaphase spreads or interphase nuclei preparedaccording to standard techniques.

The sample may also be isolated nucleic acids immobilized on a solidsurface (e.g., nitrocellulose) for use in Southern or dot blothybridizations and the like. In some embodiments, the probe may be amember of an array of nucleic acids as described, for instance, in WO96/17958. In some cases, the nucleic acids may be amplified usingstandard techniques such as PCR, prior to the hybridization. The sampleis typically taken from a patient suspected of having cancer associatedwith the abnormality being detected. “Nucleic acid” refers to adeoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, and unless otherwise limited, would encompassknown analogs of natural nucleotides that can function in a similarmanner as naturally occurring nucleotides.

“Subsequence” refers to a sequence of nucleic acids that comprise a partof a longer sequence of nucleic acids.

A “probe” or a “nucleic acid probe”, as used herein, is defined to be acollection of one or more nucleic acid fragments whose hybridization toa target can be detected. The probe is typically labeled as describedbelow so that its binding to the target can be detected. In someembodiments, the sample comprising the target nucleic acid is labeledand the probe is not labeled. For instance, when the probes are preparedas an array of nucleic acids which selectively bind a number of desiredtarget sequences.

The probe is produced from a source of nucleic acids from one or moreparticular (preselected) portions of the genome, for example one or moreclones, an isolated whole chromosome or chromosome fragment, or acollection of polymerase chain reaction (PCR) amplification products.The probes of the present invention are produced from nucleic acidsfound in the regions of genetic alteration as described herein. Theprobe may be processed in some manner, for example, by blocking orremoval of repetitive nucleic acids or enrichment with unique nucleicacids. Thus the word “probe” may be used herein to refer not only to thedetectable nucleic acids, but to the detectable nucleic acids in theform in which they are applied to the target, for example, with theblocking nucleic acids, etc. The blocking nucleic acid may also bereferred to separately. What “probe” refers to specifically is clearfrom the context in which the word is used.

“Hybridizing” refers the binding of two single stranded nucleic acidsvia complementary base pairing.

“Bind(s) substantially” or “binds specifically” or “binds selectively”or “hybridizing specifically to” refers to complementary hybridizationbetween a probe and a target sequence and embraces minor mismatches thatcan be accommodated by reducing the stringency of the hybridizationmedia to achieve the desired detection of the target nucleic acidsequence. These terms also refer to the binding, duplexing, orhybridizing of a molecule only to a particular nucleotide sequence understringent conditions when that sequence is present in a complex mixture(e.g., total cellular) DNA or RNA. The term “stringent conditions”refers to conditions under which a probe will hybridize to its targetsubsequence, but to no other sequences. Stringent conditions aresequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures.Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (Tm) for the specific sequence at adefined ionic strength and pH. The Tm is the temperature (under definedionic strength, pH, and nucleic acid concentration) at which 50% of theprobes complementary to the target sequence hybridize to the targetsequence at equilibrium. Typically, stringent conditions will be thosein which the salt concentration is at least about 0.02 Na ionconcentration (or other salts) at pH 7.0 to 8.3 and the temperature isat least about 60° C. Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide.

One of skill will recognize that the precise sequence of the particularprobes described herein can be modified to a certain degree to produceprobes that are “substantially identical” to the disclosed probes, butretain the ability to bind substantially to the target sequences. Suchmodifications are specifically covered by reference to the individualprobes herein. The term “substantial identity” of nucleic acid sequencesmeans that a nucleic acid comprises a sequence that has at least 90%sequence identity, more preferably at least 95%, compared to a referencesequence using the methods described below using standard parameters.

Two nucleic acid sequences are said to be “identical” if the sequence ofnucleotides in the two sequences is the same when aligned for maximumcorrespondence as described below. The term “complementary to” is usedherein to mean that the complementary sequence is identical to all or aportion of a reference nucleic acid sequence.

Sequence comparisons between two (or more) nucleic acids are typicallyperformed by comparing sequences of the two sequences over a “comparisonwindow” to identify and compare local regions of sequence similarity. A“comparison window”, as used herein, refers to a segment of at leastabout 20 contiguous positions, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted by thelocal homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482(1981), by the homology alignment algorithm of Needleman and Wunsch J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearsonand Lipman Proc. Nati. Acad. Sci. (U.S.A.) 85: 2444 (1988), bycomputerized implementations of these algorithms.

“Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the nucleic acid sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison and multiplyingthe result by 100 to yield the percentage of sequence identity.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to the same sequence under stringentconditions. Stringent conditions are sequence dependent and will bedifferent in different circumstances. Generally, stringent conditionsare selected to be about 5° C. lower than the thermal melting point (Tm)for the specific sequence at a defined ionic strength and pH. The Tm isthe temperature (under defined ionic strength and pH) at which 50% ofthe target sequence hybridizes to a perfectly matched probe. Typically,stringent conditions will be those as described above.

As used herein, an “antibody” refers to a protein consisting of one ormore polypeptides substantially encoded by immunoglobulin genes orfragments of immunoglobulin genes. The recognized immunoglobulin genesinclude the kappa, lambda, alpha, gamma, delta, epsilon and mu constantregion genes, as well as the myriad immunoglobulin variable regiongenes. Light chains are classified as either kappa or lambda. Heavychains are classified as gamma, mu, alpha, delta, or epsilon, which inturn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,respectively.

The phrase “specifically binds to a protein” or “specificallyimmunoreactive with”, when referring to an antibody refers to a bindingreaction which is determinative of the presence of the protein in thepresence of a heterogeneous population of proteins and other biologics.Thus, under designated immunoassay conditions, the specified antibodiesbind to a particular protein and do not bind in a significant amount toother proteins present in the sample. Specific binding to a proteinunder such conditions may require an antibody that is selected for itsspecificity for a particular protein. For example, antibodies can beraised to the particular proteins disclosed here. Such antibodies willbind the proteins and not any other proteins present in a biologicalsample. A variety of immunoassay formats may be used to selectantibodies specifically immunoreactive with a particular protein. Forexample, solid-phase ELISA immunoassays are routinely used to selectmonoclonal antibodies specifically immunoreactive with a protein. SeeHarlow and Lane (1988) Antibodies, A Laboratory Manual, Cold SpringHarbor Publications, New York, for a description of immnunoassay formatsand conditions that can be used to determine specific immunoreactivity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a physical map of P1 probes along chromosome 3. The P1cloness were picked using STSs of known genetic location alongchromosome 3. P1 clones corresponding to genes were picked using PCRprimers specific to each gene.

FIG. 2 shows clone order as used in this study. The vertical bars arethe locations of the STS's overlapping the YAC and P1 clones, confirmedby PCR analysis. Clone order in these studies is confirmed further fromGenethon and Whitehead data.

FIG. 3 shows ratios of green (test probe) to red (reference probe)signals as a function of probe position along the chromosome in ovariancancer cell lines, breast cancer cell lines, and melanoma cell lines.

FIG. 4 shows relative copy number ratios in ovarian tumor samples.

DESCRIPTION OF THE PREFERRED EMBODIMENT

Identification of chromosomal regions and genes associated with cancerThe present invention is based on a comprehensive molecular cytogeneticanalysis of the genomes of ovarian cancer cells using comparativegenomic hybridization (CGH). CGH studies on epithelial ovarian cancerhave revealed several regions that are present in increased or decreasedcopy number. More than 40% of these tumors show an increase in copynumber on the long arm of chromosome 3, in particular in the region of3q25-3qter (Iwabuchi et al., supra). This increase in copy number seemsto be an early event for ovarian cancer. Increases in copy number inthis region have also been observed in brain tumors, lung cancer, headand neck tumors, renal cell and other urinary tumors, cervical cancer,and ductal breast cancer.

Genomic regions that are found to be sites of increased DNA copy numberin a large fraction of the cell lines and primary tumor cells are likelyto include oncogenes that are present at increased copy number and henceoverexpressed. Gene amplification is one method by which cells escapefrom normal controls of proliferation. The resulting overexpression oraltered expression of these genes and their products is believed to playan important role in the development of a variety of human cancers(Weinberg, Cancer 61:1963-1968 (1988); Bishop, Cell 64: 235-248 (1991)).

The present invention is based in part on the discovery of specificcloned genomic DNA sequences showing increased copy number in a 2 MBregion at 3q26.3 region. Increased copy number was assessed using FISHand a number of P1, YAC, and cosmid clones known to map to this region.As shown below, one of the P1 clones and 5 YAC clones associated withthe region have shown an increase in copy number in 5 out of 5 ovariancancer cell line and 6 out of 6 tumor samples tested. The P1 was pickedusing PCR primers specific to the Glucose transporter gene, GLUT2. Thisgene is responsible for glucose signaling for beta cell insulin release.Its RNA product is found mostly in adult liver and pancreas,specifically in insulin-producing beta cells (Fukumoto et al., Proc.Nat. Acad. Sci. 85:5434-5438 (1988)). The sequence of cDNA from the geneis described in Fukumoto et al. This gene has been associated with isnon-insulin-dependent diabetes mellitus (NIDDM). In NIDDM the highlyconserved regions of this gene have been found mutated, resulting inabolished transport activity of the gene (Mueckler et al., J. Biol.Chem. 269:17765-17767 (1994)).

The 3q26 region also harbors the sequences for another gene, thecatalytic subunit of phosphatidylinositol kinase type 3 (P13K). Thecloning of cDNA and genomic DNA encoding the catalytic subunit isdescribed in Volinia et al. Genomics 24:472-477 (1994) and WO93/21328.

P13K is a heterodimeric enzyme comprising a 110-kDa cataiytic subunit(P1K3CA) and an 85 kD regulatory subunit that binds to tyrosinephosphopeptide sites linked to receptors serving diverse signalfunctions. Along with its regulatory subunit, PIK3CA binds to growthfactor receptors or associated signal complex proteins after ligandinduced activation, and phosphorylates phosphoinositides. The lipidproducts generated by P13K accumulate in cells activated by growthfactors (Parker and Waterfield, Cell Growth and Differentiation3:747-752 (1992)) or in cells transformed by the polyoma middle Tantigen (Serunian et al., J. Virol. 64:4718-4/25 (1990)). P13K is alsoassociated with activated src (Fukui and Hanafusa, Mol. Cell. Biol.9:1651-1658 (1989)). Relatively little is known about the mechanism ofsignal transduction for P13K. A proto-oncogene product, theserine/threonine kinase known as protein kinase B (PKB, also known asAKT and Rac), has been placed downstream of P13K (see, Burgering andCoffer, Nature 376:599-602 (1995); Franke et al., Cell 81:727-736(1995)). The AKT2 oncogene has itself shown amplification at the DNA andRNA level in ovarian cancer samples (Bellacosa et al., Int. J. Cancer64:280-285 (1995)).

In addition, P13K activity is required to maintain basal and insulinstimulated glucose and amino acid transport (Tsakiridis et al.,Endocrinology 136:4315-4322 (1995)). It is therefore likely that anincreased expression in P13K levels could also upregulate the nearbyGLUT2 gene. Compounds that inhibit expression of GLUT2 or inhibitactivity of the protein have therapeutic potential in cancers, such asovarian cancer. A number of glucose transport inhibitors are available.Exemplary glucose transport inhibitors include cytochalasin B andethanol (see, e.g., Colville et al. Biochemical Journal 290:701-706(1993) and Nagainatsu et al., Bioch. Molec. Biol. Int. 37:675-680(1995)).

A number of high molecular weight kinases have been cloned that havesequence similarities to P1K3CA. These kinases have a range of cellularfunctions such as meiotic and V(D)j recombination, chromosomemaintenance and repair, cell cycle progression, and cell cyclecheckpoints, and with dysfunctions resulting in medical disordersranging from a loss of immunological function to cancer. Therefore,increases in copy number in the P1K3CA in ovarian tumor samples may haveimplications in the level of tumor aggressiveness or patient prognosis,and the analysis of this gene at the tumor level could improve earlydiagnosis, and assist in better patient therapy and survival for thisdisease.

In some embodiments of the invention, probes specific to 19q13.1-13.2can be used in the methods, as well. Amplification of this region hasbeen correlated with ovarian cancer using FISH and molecular studies(see, e.g., Thompson et al. Cancer Genet Cytogenet 87: 55-62 (1996)).The AKT2 gene, discussed above, is located in this region. AKT2 encodesa member of a subfamily of protein-serine/threonine kinases and isthought to be a human homologue of an oncogene isolated from theretrovirus, AKT8. Staal, Proc Natl Acad Sci U S A 84:5034-7 (1987). AcDNA encoding the protein is described by Cheng et al. Proc Natl AcadSci USA 89: 9267-71 (1992). Amplification of 19q13.1-13.2 region andoverexpression of the AKT2 gene have been identified in ovarian andpancreatic cancer (see, e.g., Bellacosa et al., supra, Thompson et al.supra, and Miwa et al. Biochem Biophys Res Commun 225:968-74 (1996)).Inhibition of AKT2 expression and tumorigenicity has been demonstratedusing antisense RNA. Cheng et al. Proc Natl Acad Sci USA 93:3636-41(1996).

AKT activity appears to be regulated by binding ofphosphatidylinositol-3,4-biphosphate (Ptdins-3,4-P₂) to a pleckstrinhomology domain. The activation of AKT has been associated withincreased cell survival through a reduction in apoptosis. Withoutwishing to be bound by theory, it is believed that amplification ofPIK3CA in ovarian cancer contributes to cancer progression and/orinitiation by reducing apoptotic death and increasing cell proliferationrate. The possible decrease in apoptosis is relevant since apoptosislikely plays an important role removal of epithelial cells that becomedetached from the stroma during ovulation. Reduced apoptosis in thesecells might lead to malignancy since severed studies now suggest thatdisruption of the stroma (e.g. by overexpression of metalloproteinases)causes cancer in murine mammary cells.

In the present invention it has been found that both the PIK3CA and AKT2genes are amplified and overexpressed in cancers, such as ovariancancer. Thus, detection of amplification and/or overexpression of bothgenes is useful in the early diagnosis of cancers.

In addition, in some embodiments, the expression of other genesassociated with cancer (e.g., tumor suppressor genes or oncogenes) canbe monitored in the present invention. For instance, expression ofwild-type p53 can be monitored according to known techniques. Mutationor loss of the p53 gene is the most common genetic alteration in humancancers (Bartek etal. (1991) Oncogene, 6: 1699-1703, Hollstein et al.(1991) Science, 253: 49-53).

Preparation of Probes of the Invention

A number of methods can be used to identify probes which hybridizespecifically to the 3q26 region other than those exemplified here. Forinstance, probes can be generated by the random selection of clones froma chromosome specific library, and then mapped to each chromosome orregion by digital imaging microscopy. This procedure is described inU.S. Pat. No. 5,472,842. Briefly, a genomic or chromosome specific DNAis digested with restriction enzymes or mechanically sheared to give DNAsequences of at least about 20 kb and more preferably about 40 kb to 300kb. Techniques of partial sequence digestion are well known in the art.See, for example Perbal, A Practical Guide to Molecular Cloning 2nd Ed.,Wiley N.Y. (1988). The resulting sequences are ligated with a vector andintroduced into the appropriate host. Exemplary vectors suitable forthis purpose include cosmids, yeast artificial chromosomes (YACs),bacterial artificial chromosomes (BACs) and P1 phage. Typically, cosmidlibraries are prepared. Various libraries spanning entire chromosomesare also available commercially from for instance Genome Systems.

Once a probe library is constructed, a subset of the probes isphysically mapped on the selected chromosome. FISH and digital imageanalysis can be used to localize clones along the desired chromosome.Briefly, the clones are mapped by FISH to metaphase spreads from normalcells using e.g., FITC as the fluorophore. The chromosomes may becounterstained by a stain which stains DNA irrespective of basecomposition (e.g., propidium iodide), to define the outlining of thechromosome. The stained metaphases are imaged in a fluorescencemicroscope with a polychromatic beam-splitter to avoid color-dependentimage shifts. The different color images are acquired with a CCD cameraand the digitized images are stored in a computer. A computer program isthen used to calculate the chromosome axis, project the two (for singlecopy sequences) FITC signals perpendicularly onto this axis, andcalculate the average fractional length from a defined position,typically the p-telomere. This approach is described, for instance, inU.S. Pat. No. 5,472,842.

Sequence information of the genes identified here permits the design ofhighly specific hybridization probes or amplification primers suitablefor detection of target sequences from these genes. As noted above, thecomplete sequence of these genes is known. Means for detecting specificDNA sequences within genes are well known to those of skill in the art.For instance, oligonucleotide probes chosen to be complementary to aselected subsequence within the gene can be used. Alternatively,sequences or subsequences may be amplified by a variety of DNAamplification techniques (for example via polymerase chain reaction,ligase chain reaction, transcription amplification, etc.) prior todetection using a probe. Amplification of DNA increases sensitivity ofthe assay by providing more copies of possible target subsequences. Inaddition, by using labeled primers in the amplification process, the DNAsequences may be labeled as they are amplified.

Labeling Probes

Methods of labeling nucleic acids are well known to those of skill inthe art. Preferred labels are those that are suitable for use in in situhybridization. The nucleic acid probes may be detectably labeled priorto the hybridization reaction. Alternatively, a detectable label whichbinds to the hybridization product may be used. Such detectable labelsinclude any material having a detectable physical or chemical propertyand have been well-developed in the field of immunoassays.

As used herein, a “label” is any composition detectable byspectroscopic, photochemical, biochemical, immunochemical, or chemicalmeans. Useful labels in the present invention include radioactive labels(e.g. ³²P, ¹²⁵I, ¹⁴C, ³H, and ³⁵S), fluorescent dyes (e.g. fluorescein,rhodamine, Texas Red, etc.), electron-dense reagents (e.g. gold),enzymes (as commonly used in an ELISA), colorimetric labels (e.g.colloidal gold), magnetic labels (e.g. Dynabeads™), and the like.Examples of labels which are not directly detected but are detectedthrough the use of directly detectable label include biotin anddioxigenin as well as haptens and proteins for which labeled antisera ormonoclonal antibodies are available.

The particular label used is not critical to the present invention, solong as it does not interfere with the in situ hybridization of thestain. However, stains directly labeled with fluorescent labels (e.g.fluorescein-12-dUTP, Texas Red-5-dUTP, etc.) are preferred forchromosome hybridization.

A direct labeled probe, as used herein, is a probe to which a detectablelabel is attached. Because the direct label is already attached to theprobe, no subsequent steps are required to associate the probe with thedetectable label. In contrast, an indirect labeled probe is one whichbears a moiety to which a detectable label is subsequently bound,typically after the probe is hybridized with the target nucleic acid.

In addition the label must be detectible in as low copy number aspossible thereby maximizing the sensitivity of the assay and yet bedetectible above any background signal. Finally, a label must be chosenthat provides a highly localized signal thereby providing a high degreeof spatial resolution when physically mapping the stain against thechromosome. Particularly preferred fluorescent labels includefluorescein-12-dUTP and Texas Red-5-dUTP.

The labels may be coupled to the probes in a variety of means known tothose of skill in the art. In some embodiments the nucleic acid probesare labeled using nick translation or random primer extension (Rigby, etal. J. Mol. Biol., 113: 237 (1977) or Sambrook et al., MolecularCloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. (1989)). Particularly preferred methods for labeling probesare described in U.S. Pat. No. 5,491,224. These methods involve directlabeling the probes by chemical modification of cytosine residues.

Use of Nucleic Acids of the Invention to Detect Chromosomal Alterations

Using the results provided here, one of skill can prepare nucleic acidprobes specific to the 3q26 region of genetic alteration that isassociated with ovarian and other cancer. In particular, nucleic acidsequences from the GLUT2 gene or the PIK3CA gene can be used to detectcopy number increase of these genes. The probes can be used in a varietyof nucleic acid hybridization assays to detect the presence (inparticular increased copy number) of the target gene. Thus, the probesare useful, for example, in the early diagnosis or prognosis of cancer.As noted above, the probes are particularly useful for detectingalteration associated with ovarian cancer. The regions can also be usedfor a large number of other cancers as described above.

The genetic alterations are detected through the hybridization of aprobe of this invention to a nucleic acid sample in which it is desiredto screen for the alteration. Suitable hybridization formats are wellknown to those of skill in the art and include, but are not limited to,variations of Southern Blots, northern blots, CGH, in situ hybridizationand quantitative amplification methods such as quantitative PCR (see,e.g. Sambrook et al., Kallioniemi et al., Proc. Natl Acad Sci USA, 89:5321-5325 (1992), and PCR Protocols, A Guide to Methods andApplications, Innis et al., Academic Press, Inc. N.Y., (1990)).

The sample used in the methods will, of course, depend upon theparticular method used to detect the target. For instance, the nucleicacid sample can be a tissue or cell sample prepared for standard in situhybridization methods. The sample or probes may also be isolated nucleicacids immobilized on a solid surface (e.g., nitrocellulose) for use inSouthern or dot blot hybridizations and the like. In some embodiments,the probes of the invention may comprise an array of nucleic acids asdescribed, for instance, in WO 96/17958).

In a preferred embodiment, the regions disclosed here are identifiedusing in situ hybridization. Generally, in situ hybridization comprisesthe following major steps: (1) fixation of tissue or biologicalstructure to analyzed; (2) prehybridization treatment of the biologicalstructure to increase accessibility of target DNA, and to reducenonspecific binding; (3) hybridization of the mixture of nucleic acidsto the nucleic acid in the biological structure or tissue; (4)posthybridization washes to remove nucleic acid fragments not bound inthe hybridization and (5) detection of the hybridized nucleic acidfragments. The reagent used in each of these steps and their conditionsfor use vary depending on the particular application.

In some applications it is necessary to block the hybridization capacityof repetitive sequences. In this case, human genomic DNA or Cot1 DNA isused as an agent to block such hybridization. The preferred size rangeis from about 200 bp to about 1000 bases, more preferably between about400 to about 800 bp for double stranded, nick translated nucleic acids.

Hybridization protocols for the particular applications disclosed hereare described in Pinkel et al. Proc. Nati. Acad. Sci. USA, 85: 9138-9142(1988) and in EPO Pub. No. 430,402. Suitable hybridization protocols canalso be found in Methods in Molecular Biology Vol. 33: In SituHybridization Protocols, K. H. A. Choo, ed., Humana Press, Totowa, N.J.(1994). In a particularly preferred embodiment, the hybridizationprotocol of Kallioniemi et al., Proc. Natl Acad Sci USA, 89: 5321-5325(1992) is used.

Typically, it is desirable to use dual color FISH, in which two probesare utilized, each labeled by a different fluorescent dye. A test probethat hybridizes to the region of interest is labeled with one dye, and acontrol probe that hybridizes to a different region is labeled with asecond dye. A nucleic acid that hybridizes to a stable portion of thechromosome of interest, such as the centromere region, is often mostuseful as the control probe. In this way, differences between efficiencyof hybridization from sample to sample can be accounted for.

The FISH methods for detecting chromosomal abnormalities can beperformed on nanogram quantities of the subject nucleic acids. Paraffinembedded tumor sections can be used, as can fresh or frozen material.Because FISH can be applied to the limited material, touch preparationsprepared from uncultured primary tumors can also be used (see, e.g.,Kallioniemi, A. et al., Cytogenet. Cell Genet. 60: 190-193 (1992)). Forinstance, small biopsy tissue samples from tumors can be used for touchpreparations (see, e.,g., Kallioniemi, A. et al., Cytogenet. Cell Genet.60: 190-193 (1992)). Small numbers of cells obtained from aspirationbiopsy or cells in bodily fluids (e.g., blood, urine, sputum and thelike) can also be analyzed.

In various blot formats (e.g., dot blots, Southern blots, and Northernblots) nucleic acids (e.g., genomic DNA, cDNA or RNA) are hybridized toa probe specific for the target region. Either the probe or the targetcan be immobilized on the solid surface. Comparison of the intensity ofthe hybridization signal from the probe for the target region with thesignal from a probe directed to a control (non amplified or deleted)such as centromeric DNA, provides an estimate of the relative copynumber of the target nucleic acid. Procedures for carrying out Southernhybridizations are well known to those of skill in the art. see, e.g.,Sambrook et al., supra.

Detection of Proteins of the Invention

The gene products described here, GLUT2 and P1 K3CA, can be detected andquantified by any of a number of means well known to those of skill inthe art. These may include analytic biochemical methods such aselectrophoresis, capillary electrophoresis, high performance liquidchromatography (HPLC), thin layer chromatography (TLC), hyperdiffusionchromatography, and the like, or various immunological methods such asfluid or gel precipitin reactions, immunodiffusion (single or double),immunoelectrophoresis, radioimmunoassay(RIA), enzyme-linkedimmunosorbent assays (ELISAs), immunofluorescent assays, westernblotting, and the like.

In a preferred embodiment, the proteins are detected using animmunoassay. As used herein, an immunoassay is an assay that utilizes anantibody to specifically bind to the analyte (e.g., GLUT2 or PIK3CAproteins). The immunoassay is thus characterized by detection ofspecific binding of the protein to an antibody raised against it asopposed to the use of other physical or chemical properties to isolate,target, and quantify the analyte (see, e.g., U.S. Pat. Nos. 4,366,241;4,376,110; 4,517,288; and 4,837,168). For a review of the generalimmunoassays, see also Methods in Cell Biology Volume 37. Antibodies inCell Biology, Asai, ed. Academic Press, Inc. New York (1993); Basic andClinical lnmunology 7th Edition, Stites & Terr, eds. (1991).

The proteins are preferably quantified in a biological sample derivedfrom a mammal, more preferably from a human patient. As used herein, abiological sample is a sample of biological tissue or fluid thatcontains a protein concentration that may be correlated withamplification of the 3q regions disclosed here. Particularly preferredbiological samples include, but are not limited to biological fluidssuch as blood or urine, or tissue samples including, but not limited totissue biopsy (e.g., needle biopsy) samples.

The antibody (e.g., anti-GLUT2 or anti-PIK3CA) may be produced by any ofa number of means well known to those of skill in the art (see, e.g.Methods in Cell Biology Volume 37: Antibodies in Cell Biolgy, Asai, ed.Academic Press, Inc. New York (1993); and Basic and Clinical Immunology7th Edition, Stites & Terr, eds. (1991)). The antibody may be a wholeantibody or an antibody fragment. It may be polyclonal or monoclonal,and it may be produced by challenging an organism (e.g. mouse, rat,rabbit, etc.) with one of these proteins or an epitope derivedtherefrom. Alternatively, the antibody may be produced de novo usingrecombinant DNA methodology. The antibody can also be selected from aphage display library screened against the protein (see, e.g. Vaughan etal. (1996) Nature Biotechnology, 14: 309-314 and references therein).

In other embodiments, Western blot (immunoblot) analysis is used todetect and/or quantify the presence of the proteins in the sample. Thetechnique generally comprises separating sample proteins by gelelectrophoresis on the basis of molecular weight, transferring theseparated proteins to a suitable solid support, (such as anitrocellulose filter, a nylon filter, or derivatized nylon filter), andincubating the sample with the antibodies that specifically bind thedesired protein. Other assay formats include liposome imniunoassays(LIA), which use liposomes designed to bind specific molecules (e.g.,antibodies) and release encapsulated reagents or markers. The releasedchemicals are then detected according to standard techniques (see,Monroe et al. (1986) Amer. Clin. Prod. Rev. 5:34-41).

The particular label or detectable group used in an immunoassay is not acritical aspect of the invention, so long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and, in general, most any label useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include magnetic beads (e..g. Dynabeads™),fluorescent dyes (e.g., fluorescein isothiocyanate, texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and calorimetric labels such ascolloidal gold or colored glass or plastic (e.g. polystyrene,polypropylene, latex, etc.) beads.

Kits Containing Probes of the Invention

This invention also provides diagnostic kits for the detection ofchromosomal abnormalities at the regions disclosed here. In a preferredembodiment, the kits include one or more probes to the regions describedherein. The kits can additionally include blocking probes, instructionalmaterials describing how to use the kit contents in detecting thealterations. The kits may also include one or more of the following:various labels or labeling agents to facilitate the detection of theprobes, reagents for the hybridization including buffers, a metaphasespread, bovine serum albumin (BSA) and other blocking agents, samplingdevices including fine needles, swabs, aspirators and the like, positiveand negative hybridization controls and so forth.

EXAMPLES Example 1

This example describes the identification of genes in a 2 MB region at3q26.3 region. Increased copy number of this region is correlated withovarian cancer.

CGH studies have identified DNA copy number abnormalities at 3q25-26associated with ovarian tumors (Iwabuchi et al., Cancer Research55:6172-8180 (1995)). To physically map the 3q26 region, a number ofyeast artificial chromosome (YAC), P1, and cosmid clones that were knownto genetically map to the region were physically mapped to the 3qregion. Several yeast artificial chromosome (YAC) and P1 clones known togenetically map to this region were physically mapped using FISH andfractional length analysis. Clone positions were reconfirmed by PCRusing STSs specific to the region. Clones were then picked according totheir physical map position and hybridized onto interphase cells ofovarian cancer, breast cancer, and melanoma cell lines, as well asnuclei of paraffin-embedded ovarian tumors. One of the P1 clones and its5 associated YAC clones have shown increases in copy number in 5 out of5 ovarian cancer cell lines and 6 out of 6 primary ovarian tumorsamples, small increases in copy number in the breast cancer lines, andno increases in copy number in the melanoma lines. Based on theseresults, the region of increased copy number was narrowed to a 2 MBregion at 3q26.3.

Materials and Methods

Probes. Yeast artificial chromosome (YAC) clones were obtained fromGenethon/CEPH of France. YAC clones were chosen based on their geneticmap along 3q24-3qter. Each YAC was grown and checked for chimerism byFISH. P1 clones were obtained by screening a human genomic P1 library(DuPont, Boston, Mass.) using PCR with primers specific to chromosome 3.Those P1 clones mapping to 3q25-3qter were used for further study. A P1clone mapping to the 3p region was used throughout the experiments as areference marker.

Nonchimeric YAC clones as well as all the P1 clones were mapped ontochromosome 3 by digital image analysis of their physical distance fromthe terminus of the p arm (Flpter analysis) generally as described inMascio, et al. Cytometry 19:51-9 (1995) and Sakamoto et al, Cytometry,19:60-9 (1995)).

All probes were labeled for hybridization by random priming (BioPrimekit, BRL). The 3q region probes were labeled with digoxigenin-11-dUTP(Boehringer-Mannheim) and detected using Fluorescein-antidigoxigenin.The reference P1 probe on 3p was directly labeled with Texas-Red dUTP(NEN DuPont).

Normal human metaphase spreads, cell lines, and paraffin-embedded tumorsamples. Normal human metaphase spreads were prepared as previouslydescribed (Kallioniemi et al., supra). Slides were denatured in 70%formamide/2×SSC at 72 degrees for 3 to 10 minutes (depending on theslide batch) and then serially dried in 70%, 85%, and 100% ethanol.

Cell pellets of lines SKOV3, CAOV3, and OVCAR3 were obtained from ATCC.Cell pellets of lines G93, G95, 355, and 457 were kindly provided by Dr.Taetle (University of Arizona). All cells were resuspended in 2 ml of0.075M KC1 hypotonic solution, incubated at 37 degrees for 20 minutes,fixed, and dropped onto slides.

Paraffin-embedded epithelial ovarian tumor samples were provided by Dr.Teresa Yang-Feng (Yale University). All samples were checked tocontain >60% tumor cells. These samples were deparaffinized, usingxylene, washed with ethanol, then with water, digested with pepsin, andcytospun onto slides in order to concentrate the cell population.

FISH, physical mapping, and slide scorings. Cell line and normalmetaphase slides were denatured in 70% formamide/2×SSC for 5 minutes at72 degrees, followed by drying through 70%, 85%, and 100% ethanol.Paraffin-embedded tumor materials were fixed for 10 minutes inmethanol-acetic acid (3:1) prior to denaturation in 70% formamide/2×SSCfor 10 minutes at 80 degrees, then digested with 5 ug/MI proteinase Kfor 10 minutes at 37 degrees, followed by drying through 70%, 85%, and100% ethanol for 2 minutes each.

40 ng of each probe was placed on each slide along with 5 ug Cot1 DNA(to suppress repetitive sequences) in a total of 10 ul of 50%formamide/2×SSC/10% dextran sulfate, and slides were coverslipped andsealed. After an overnight incubation at 37 degrees, slides were washedto remove unbound probes, stained immunochemically withfluoresceinantidigoxigenin, counterstained with 0.2 uM4,6-diamino-2-phenylindole in antifade solution for chromosomeidentification, and visualized under fluorescent microscope. Forphysical mapping, multicolor images of metaphase chromosomes and theirassociated probes were acquired using the QUIPS (quantitative imageprocessing system). Analysis of the hybridization signals is completelyautomated, and carried out using the Xquips software (Mascio, et al.Cytometry 19:51-9 (1995) and Sakamoto et al, Cytometry 19:60-9 (1995)).Briefly, analysis consisted of chromosome segmentation, medial axiscalculation, hybridization domain segmentation, center of masscalculation, and contrast enhancement. Fractional location of a domainwas determined from the end of the short arm to the valid hybridizationsignal (Flpter analysis). On average, 20 Flpter measurements were madefor each probe, and probe location on a chromosome was reported as themean +/− one standard error of the mean of the measurements. Probe orderwas determined from the mean Flpter values.

For interphase cells, simultaneous Texas Red and Fluorescein signalswere visualized using a double bandpass filter on the X63 objective of aZeiss Axioscope camera. At least 100 cells were counted for each probeset.

Results

Physical mapping. FIG. 1 shows the order of all the P1s mapped alongchromosome 3. In addition, the order of the clones for the region ofinterest was also checked by PCR using several STSs known to map to thisregion and confirmed by comparing these results to the recentlypublished YAC maps in the Genome Directory Naylor et al., Cytogenet.Cell Genet. 72: 255-70 (1995) and the Whitehead institute's integratedmap Dib et al., Nature 380:152-4 (1996). FIG. 2 shows the order of theclones as used in this study.

Copy number abnormalities in cancer cell lines for the 3q26 region. CGHstudies delineated the region of increase in copy number at best toabout 10 Megabases. FISH with well-mapped clones specific to the regionwas used to refine the region of increase in copy number on 3q26 inovarian cancer to 2 megabases. FIG. 3 shows the data from hybridizationexperiments onto ovarian, breast, and melanoma cell lines. The graphsare relative copy numbers of the probes in the cell lines as a functionof probe distance along the chromosome. One P1 clone, Glut2, and 5 YACclones that share sequences with this P1 (683F10, 784H12, 806D8, 822G9,945H6) consistently show increases in copy number in all ovarian celllines. OVCAR3 shows a larger region of amplification throughout theregion, with the aforementioned clones still manifesting the largestincrease in copy number. Breast cancer lines ZR75-30 and mda453 showed asmaller increase in copy number for the same clones in the region, andmelanoma lines 355 and 457 failed to show increases in copy number inthis region.

Copy number abnormalities in ovarian tumor samples. Allparaffin-embedded ovarian tumor samples also show the same regions ofincrease in copy umber that were seen in the ovarian cancer cell lines.As seen from FIG. 4, the region of increase in copy number is betterdefined in the tumor samples, with a sharp increase in the relative copynumber for the P1 and its associated YACS. Increases in copy number fortumor sample 595-7615 seem to involve a larger amplicon, as all theprobes tested in this tumor show a relatively elevated copy number.

Based on the FISH results of tumor samples and cell lines, we havedelineated the critical region of increase in copy number in ovariancancer on the long arm of chromosome 3 to the region of 3q26.3 ,spanning one P1 and 5 YACs that share sequences with this P1.

The above examples are provided to illustrate the invention but not tolimit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, patents, and patent applicationscited herein are hereby incorporated by reference for all purposes.

What is claimed is:
 1. A method of screening for the presence ofincreased DNA copy number associated with cancer, the method comprising:contacting a nucleic acid sample comprising genomic DNA from a humanpatient with a probe which binds selectively to a target nucleic acidsequence at 3q26.3, wherein the target nucleic acid sequence is in aGLUT2 gene, and the probe is contacted with the sample under conditionsin which the probe binds selectively with the target nucleic acidsequence to form a stable hybridization complex; and detecting theformation of a hybridization complex, wherein the amount ofhybridization complex is increased relative to normal, thereby detectingthe presence of DNA copy number changes associated with cancer.
 2. Themethod of claim 1, wherein the nucleic acid sample is from an ovariansample from the patient.
 3. The method of claim 1, wherein the probe isa member of an array.
 4. The method of claim 1, further comprisingcontacting the sample with a reference probe which binds selectively toa centromeric DNA.
 5. The method of claim 1, wherein the step ofdetecting the hybridization complex comprises determining the copynumber of the target sequence.
 6. The method of claim 1, wherein theprobe is labeled with digoxigenin or biotin.
 7. The method of claim 1,wherein the step of detecting the hybridization complex is carried outby detecting a fluorescent label.
 8. The method of claim 7, wherein thefluorescent label is FITC.
 9. The method of claim 1, wherein the samplecomprises a metaphase cell.
 10. The method of claim 1, wherein thenucleic acid sample is from a lung sample from the patient.